Iron is an essential nutrient for virtually every organism, yet it can also be a potent cellular toxin. Dysregulated iron metabolism and iron overload are features of a growing number of human diseases. Some genes involved in cellular iron uptake and export have been identified, yet very little is known about inter- and intracellular iron transport, intracellular iron utilization, and the regulation of these processes. A combination of genetic, biochemical, and cell biological approaches is needed to understand iron metabolism and the role of iron in human disease. These approaches can be combined in the simple eukaryote, Saccharomyces cerevisiae. Studies of metal metabolism in budding yeast have yielded important insights into iron, copper, and zinc metabolism in both humans and pathogenic microorganisms. Genetic studies of iron metabolism in a simple eukaryote will allow us to discover new genes involved in iron homeostasis as well as to determine the cellular response to iron overload and iron deprivation. We have used a variety of strategies to identify genes that are involved in eukaryotic iron homeostasis. Using available genome and protein databases, we have grouped these newly identified genes into families and have begun their functional evaluation.[unreadable] We have identified and genetically characterized a novel system of eukaryotic iron uptake. Four homologous genes regulated as part of the Aft1-regulon (ARN1-4) were found to facilitate the transport of siderophores. We have determined that the ferrichrome (FC) transporter, Arn1, undergoes a distinct pattern of intracellular trafficking in response to ferrichrome. The trafficking of the Arn1p transporter suggested the presence of a high-affinity receptor for FC. Whether this receptor was a separate gene product or part of the Arn1p transporter was unknown. The ARN transporters contain a unique carboxyl-terminal domain consisting of two predicted transmembrane domains, an extracytosolic loop, and a cytosolic tail, which are not present in other MFS transporters. We have conducted a structure-function analysis of this domain to evaluate its role in FC binding and trafficking. Mutations in the extracytosolic loop indicate that it functions as a receptor domain, and the binding of FC to this domain controls the sorting of the transporter. Thus, for this simple eukaryote, FC receptor and transporter functions have been combined in a single gene product. We have further evaluated the role of posttranslational modifications in control of the trafficking of Arn1p. Arn1p is modified by poly-ubiquitin chains attached primarily at a single lysine residue on the amino terminus. Ubiquitination occurs via the E3 ligase Rsp5, and the attachment of ubiquitin chains controls multiple steps in the intracellular trafficking of Arn1p.[unreadable] Our microarray analysis unexpectedly revealed that other metabolic pathways are regulated in response to iron availability. The iron-dependent enzyme glutamate synthetase is also transcriptionally down-regulated in response to iron deprivation, and we have characterized the transcriptional control regions of this gene to identify the iron-responsive sequences. To identify the transcription factor that controls the iron-dependent activation of GLT1, we carried out a screen for genes that stimulate GLT1 expression under conditions of iron deprivation. This strategy allowed us to identify the iron-dependent transcriptional regulator UGA3, and further studies have revealed that iron controls a gamma-amino butyric acid recycling pathway in yeast. This pathway, known as the ?GABA shunt? in plants, has not been characterized in fungi, but appears to constitute an important metabolic adaptation to elevated iron levels in yeast. Microarray studies are underway to determine what other metabolic pathways are regulated by Uga3 in yeast. [unreadable] Very little is known about heme transport in eukaryotes and no fungal heme transporters have been identified. We have identified a gene family from C. albicans and S. cerevisiae that, when overexpressed in S. cerevisiae, facilitates the uptake of heme. Members of this family of genes all localize to the endoplasmic reticulum and serve an essential function in yeast. Surprisingly, we have obtained genetic and biochemical evidence that this family of genes encodes a novel family of flavin adenine dinucleotide carriers that facilitate the process of FAD transport into the endoplasmic reticulum, where it is required for oxidative protein folding. As this transport activity is required for all eukaryotes, studies are under way to identify the orthologous genes in multicellular eukaryotes. [unreadable] Although S. cerevisiae does not take up heme in response to iron deprivation, we have determined that this species does have an inducible heme uptake system that is activated under heme deficiency and hypoxic conditions. Microarray analysis of strain grown under these conditions indicates that a number of putative transporters are transcriptionally activated. Studies are underway to determine if heme uptake is dependent on the expression of any of these putative transporters.